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Therapeutic targets are typically identified using in vitro approaches, but the complex interactions of migratory immune cells with many other cell populations are very difficult to model using in vitro systems. The Wucherpfennig Lab developed an in vivo shRNA screening approach that enables discovery of negative regulators of cytotoxic T cell function in tumors (Nature 2014, 506: 52-57).
The study took advantage of pooled shRNA screening approaches with which many genes can be interrogated simultaneously. The goal was to discover genes that inhibit T cell function in immunosuppressive tumor microenvironments. When T cells recognize a microbial antigen in the setting of an acute infection, T cells undergo a fundamental change in cellular state, switching from quiescence to rapid cell division and acquisition of effector functions. Such T cell receptor-driven proliferation is inhibited in tumors by a variety of negative signals. The Wucherpfennig lab asked which genes encode key negative regulators of T cell function in tumors by screening for shRNAs that restored T cell proliferation following tumor antigen recognition.
CD8 T cells were thus infected with lentiviral pool shRNA libraries following brief in vitro culture with homeostatic cytokines. CD8 T cells (specific for ovalbumin) were injected into mice bearing B16 tumors that expressed ovalbumin as a surrogate tumor antigen. shRNAs that enabled T cell accumulation in tumors were identified by Illumina sequencing of the shRNA cassette from tissue-infiltrating T cells. This approach provided a quantitative representation of all shRNAs in the pool across several different organs (tumor, tumor-draining lymph node, irrelevant lymph nodes and spleen). A total of 43 genes were discovered, and silencing of seven of these genes increased T cell accumulation in tumors relative to spleen at least tenfold. Silencing Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases, substantially reduced T cell apoptosis in tumors and also enhanced T cell proliferation, cytokine production, and anti-tumor activity.